Biological samples, usually serum or plasma, but also urine, cerebral spinal fluid or other bodily fluids, containing immunologically active antigens often also contain quantities of antibodies that react with these antigens. These antibodies will bind to the antigens present and form immunocomplexes. These immunocomplexes may prevent detection of the antigens by tying up the binding sites present on the antigens. This poses a particular problem when a sample is being tested with an immunoassay for the presence of such antigen. Generally, the immunoassay will contain an antibody that is specific for the antigen in question, and will therefore need to have access to the binding sites on the antigen in order to capture or form an immunocomplex with it. If all of the binding sites on the antigen are complexed with antibodies present in the sample, no capture of the antigen will occur in the immunoassay, and the antigen will not be detected.
This problem has been seen in particular with the detection of the p24 core antigen of the human immunodeficiency virus, HIV-1.
In order to detect infection by HIV-1, to check on the progress of the disease it causes, acquired immunodeficiency syndrome (AIDS), or to monitor therapy, detecting the p24 core antigen of HIV-1 is highly desirable. However, available assays for the detection of HIV-1 p24 antigenemia in serum or plasma are able to detect HIV infection only before seroconversion of antibody from negative to positive occurs. Once antibodies to HIV begin to appear, these tests are severely limited in their ability to detect antigen. It is generally thought that these limitations are caused by the presence of anti-p24 antibodies in the blood that complex with the antigen, thereby blocking the reactive sites that would otherwise be available to complex with the antibodies of the test immunoassays. T. M. McHugh et al. discussed this issue in "Relation of Circulating Levels of Human Immunodeficiency Virus (HIV) Antigen, Antibody to p24, and HIV-Containing Immune Complexes in HIV-Infected Patients," J. Inf. Dis., 158, no. 5, pages 1088-1091, November 1988.
At least two solutions have been proposed to deal with this problem. Both solutions attempt to dissociate the immunocomplexes formed in the sample while still maintaining the viability of the antigen and at the same time prevent the newly dissociated antibody from recombining with the antigen.
The currently preferred solution is that of subjecting the serum or plasma sample to an acid dissociation treatment prior to testing it in an immunoassay (P. Nishanian et al., "A Simple Method for Improved Assay Demonstrates that HIV p24 Antigen is Present as Immune Complexes in Most Sera from HIV-Infected Individuals," J. Inf. Dis., 162, pages 21-28, July, 1990; R. C. Bollinger, Jr. et al., "Acid Dissociation Increases the Sensitivity of p24 Antigen Detection for the Evaluation of Antiviral Therapy and Disease Progression in Asymptomatic HIV-Infected Persons," J. Inf. Dis., 165, pages 913-916, May, 1992; S. Kontio, "Sensitive One-Step Enzyme Immunoassay for HIV-1 p24 Antigen in Human Blood Specimens and Cell Culture Supernatants," J. Imm. Methods, 139, pages 257-263, 1991). Generally, the method involves taking the serum or plasma, mixing it with an acid such as 0.5N HCl (pH from about 2.0 to 3.0), incubating for 60 to 90 minutes at 37.degree. C., and neutralizing with a base such as 0.5N NaOH (pH from about 6.8 to 7.2). The sample so prepared is then used in the immunoassay of choice.
This method relies on the supposition that under acidic conditions antibodies are irreversibly inactivated more readily than many antigens. Antigen detection in the presence of antibody has been shown to be enhanced substantially by this method, but there are several drawbacks.
The first is that antigens as well as antibodies are inactivated in this method, as demonstrated by some laboratory procedures where although the test samples are acid pretreated, the standards containing only p24 are not. It is easy to see that when the antigens are also affected during treatment, the sensitivity needed to detect antigen in low antigen containing and/or non-antibody containing samples is reduced.
Another drawback is that the test sample must be pretreated prior to its being tested in an immunoassay. This involves an additional step of handling the test sample in order to do the immunoassay, increasing the chances of infection of laboratory personnel. Exposure of laboratory personnel to possibly infectious samples increases as the number of steps to perform the immunoassay increases, as does the possibility for error when transferring the contents of one microtiter plate well to another.
Acid treatment may cause precipitation or clotting of biological samples, thereby interfering with later assay procedures. Because of this problem, the samples must be diluted, usually three fold, to reduce the effects of the precipitation or clotting. However, when a sample having only a small amount of antigen is diluted, the chances of detecting the antigen decrease.
Another method proposed to dissociate the immunocomplex while leaving the antigen intact and available for further testing is the method of S. A. Fiscus et al., using a polyethylene glycol precipitation method (S. A. Fiscus et al., "Detection of Infectious Immune Complexes in Human Immunodeficiency Virus Type 1 (HIV-1) Infections: Correlation with Plasma Viremia and CD4 Cell Counts," J. Inf. Dis., 164, pages 765-769, October 1991). This method is a complex one where the test plasma is specially filtered, mixed with polyethylene glycol (PEG-8000), vortexed, refrigerated overnight, centrifuged, and the pellets washed twice in 2% PEG. The supernatants and aliquots of the PEG precipitates can then be tested in an immunoassay that detects p24. This method appears to have detected p24 in samples containing anti-p24 antibody or p24 immunocomplexes, but it is not currently used as a standard method to detect the presence of p24. Among other drawbacks, it is an involved procedure with multiple steps necessitating much handling of the test specimen and long incubation periods.
What is needed is a method for detecting the presence of antigen that has been immunocomplexed with antibody in a test sample, in particular, p24 complexed with anti-p24 antibodies in a sample. Additionally, such method should be done with a minimum amount of steps, so as to reduce the exposure of the laboratory personnel handling potentially infected samples.